“The Achilles heel of gene therapy is gene delivery”, said by Inder M. Verma, the famous biologist of the Salk institute in San Diego, Calif., USA. While delivering an exogeneous gene into a cell, scientists must compress the corresponding deoxyribonucleic acid (DNA) into a small package absorbed by the cell. But that is not enough. The gene must be protected from cellular enzyme digestion. While the gene arrived into the nucleus, the gene must remain in an active form. For the reasons that the DNA carrying negative charges, the size of itself, and nucleases in the blood, if the naked DNA is directly administrated, the naked DNA may suffer nucleolytic degradation prior to its delivery to the target, resulting in the insufficient therapy. However, this problem can be overcome by utilizing a suitable carrier to enhance DNA into the nucleus.
Common carriers for gene therapy include viral and non-viral vectors. Common viral vectors include retroviral and adenoviral vectors. Ribonucleic acid (RNA) viruses with envelope are the most popular in retroviral vectors. The RNA viruses generate double-stranded (ds) DNA by reverse transcriptase action, and then integrate into the host chromosomes, to achieve characteristics of gene therapy and stable expression.
Viral carriers have better delivery efficiency and expression in vivo as their advantages, but there are some drawbacks existed in their potential risks. For example, it must be considered that random insertion may induce undesirably insertional mutagenesis in using viruses. There are replication competent viruses (RCV) may be generated due to gene recombination happened in packaging the viral particles, or immune responses may be induced by pathogenic viruses which are recombined by viral vectors and latent viruses. Furthermore, it is difficult to produce viral vectors by cell culturing, so it is not easy to produce viral vectors in scale.
The approaches by non-viral vectors, such as liposomes or directly injecting DNA into a cell by microinjection or a gene gun, can avoid possible risks and side effects caused by viral vectors. “Lipofection” uses some liposomes made of nucleic acids and phospholipids with no charge or polarity in various ratios to pack DNA therein, which would pass through the cell membrane and introduce DNA into a cell or tissue by endocytosis. A type of liposome, for examples, linked with a specific antibody, or a ligand of a receptor chemically linked with poly-lysine as a DNA binding domain, can improve to facilitate specificity of DNA delivered to the specific cell. However, the transfection efficiency of DNA delivered by liposomes is not efficient, and most DNA is degraded inside endosomes, so the actual DNA expression in the nucleus is limited.
On one hand, investigators try to understand and reduce the risk of viral vectors used in the gene therapy, and on the other hand, they designate alternative approaches by utilizing polymers where plasmid DNA is embedded in. Mark E. Davis of the professor and Sue J. Huang, in California Institute of Technology, USA, disclose a cationic polymer derived from β-Cyclodextrin (CD). See, Bioconjugate Chemistry (2002) 13, page 630. This kind of material, which is non-toxic, non-immunogenic, dissolved in water, and can be modified on the surface of CD particle by linking with polyethylene glycol (PEG) having adamantine, for CD/DNA nanoparticles with uniform size, and may not form aggregate with proteins in the plasma to lose its biological potential. The PEG modifies the surface of CD particles, and provides “chemical hook” to bind to other substance that can lead CD particles or delivery gene to a specific cell.
Methods of making and using poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), which is an analogue of poly-L-lysine, for delivering a gene into a cell are disclosed in U.S. Pat. No. 6,217,912. PAGA is grafted with PEG, to form block copolyines with other polymers, such as poly-L-lysine, polyarginine, polyorthithine, histidine, avidin, protamines, polylactides, or polylactic/glycolic acid, and provided complexes with nucleic acids for delivering a gene into a cell.
U.S. Pat. No. 6,083,741 and PCT Pub. No. WO96/15811 disclose a conjugate which is formed with a polylysine coupling to a integrin receptor binding moiety comprising a peptide of the sequence arginine-glycine-asparagine (RGD). The sequence RGD binds specifically integrin receptors on the tissue, and leads DNA/Polylysine complexes into cells in the target tissue.
U.S. Pat. No. 5,965,404 and T.W. Pat. No. 496,898 disclose a process of introducing nucleic acid into mammalian cells, in which combines DNA with a partial amount of polylysine of various lengths, and subsequently the rest of polylysine, in majority portion, is added into complexes, and optionally add chloroquine in the presence of ethylene glycol and/or glycerol, for introducing nucleic acids into primary cells to obtain stably transformed cells. Thereby, polycations, such as polylysine, can form complexes with DNA, or conjugates of polycations and transferrin may complexate with DNA to introduce nucleic acids into specific primary cells.
During developing these polymers, it must be considered that polymers possess biocompatibility, specificity to target tissues or cells, high efficiency of gene transfection, low immune response and so on. Additionally, polymers must be biodegradable. Polyurethane known in the prior art is modified in some suitable process, such as adding wood flour, sugars, starch, to make polyurethane more biodegradable. PCT Pub. No. WO 89/05830 is disclosed matrix materials for tissue repairing based on biodegradable polyurethanes and polyester polyol prepolymers.